丝胶蛋白源Tn和T抗原的释放制备及质谱分析

Preparative Release and Mass Spectrometric Analysis of Tn and T Antigens Existing in Sericin

以蚕茧丝胶蛋白源Tn抗原和T抗原为研究对象,采用“一釜法”、氨水催化法和链霉蛋白酶E(PronaseE)水解法对丝胶蛋白O-糖链进行释放,并通过固相萃取柱进行分级纯化,以高效液相色谱仪(HPLC)进行分离制备;利用电喷雾质谱(ESI-MS)、串联质谱(MS/MS)及在线液相色谱-质谱联用仪(Online LC-MS)进行了结构鉴定和定量分析.结果表明,丝胶蛋白中O-GalNAc含量为1.58μg/g,O-GalNAcGal含量为0.54μg/g,二者丰度比约为5.3∶1;并且氨水可高效释放出其还原性O-连接单糖GalNAc,通过液相色谱法可实现其精细分离纯化,而以等量PronaseE对丝胶蛋白水解可有效释放出Tn抗原和T抗原.

Silkworm sericin-derived Tn and T antigens,a group of biomarkers of many cancers,were targeted in this study.The O-glycans of sericin were first released using the“one-pot”non-reductive release method,the ammoniacatalyzed non-reductive release strategy and the pronase E digestion approach.The samples were subsequently fractionated for purification using solid-phase extraction columns and separated for preparation via high-performance liquid chromatography(HPLC).The generated glycan samples were finally analyzed in detail by means of electro⁃spray ionization mass spectrometry(ESI-MS),tandem mass spectrometry(MS/MS)and liquid chromatography coupling online with mass spectrometry(Online LC-MS).The obtained results showed that the O-glycans of sericin just include O-GalNAc and O-GalNAcGal,the contents of which have been determined to be 1.58μg/g and 0.54μg/g,respectively.Their molar ratio in sericin is approximately 5.3∶1.The O-linked monosaccharide of sericin,GalNAc,allows efficient non-reductive release as a reducing form in ammonium hydroxide and fine separation by liquid chromatography.Tn and T antigens can be efficiently generated from sericin using pronase E with an amount equal to the raw glycoprotein.